The U.S. Department of Energy has identified malic acid and other 1,4-dicarboxylic acids (fumaric and succinic) as building block chemicals that could be made in large quantities from renewable carbohydrates and converted to high volume products (27). Presently, malic acid usage is limited to pharmaceuticals, cosmetics, and acidulants in the food industry (2, 23). It is produced as a racemic mixture by chemical synthesis (hydration of maleic or fumaric acid) or as enantiomerically pure L-malate by the enzymatic hydration of fumarate (immobilized cells or fumarase) (2, 6, 21). Substrates for the synthesis of malic acid (maleic acid, fumaric acids, maleic anhydride) are derived from petroleum (22). Increases in oil and gas prices coupled with concerns about climate change and global warming have renewed interests in the production of malic acid by microbial fermentation (7).
Malate can be made by a wide range of microorganisms using aerobic or microaerophilic processes (Table 1) (1, 16, 18-19, 25). Aspergillus flavus is the best known producer (1). This organism can ferment glucose to malate at relatively high yield (1.28 mol malate per mol glucose), titer (113 g liter−1) and productivity (0.59 g liter−1 h−1). However, this biocatalyst is not useful in industrial processes due to the potential for aflatoxin production (1, 5). A sugar-tolerant yeast, Zygosaccharomyces rouxii, was recently found to produce 75 g liter−1 malic acid when cultured aerobically in complex medium containing 300 g liter−1 glucose (25). Malate has also been produced by engineered strains of Saccharomyces cerevisiae (20, 28). Overexpression of plasmid-born genes encoding pyruvate carboxylase, cytosolic malate dehydrogenase, and a heterologous malate transporter resulted in the production of 59 g liter−1 malate (28).
Escherichia coli has been previously engineered in our lab for the efficient production of succinate by increasing the expression of pyruvate carboxykinase, an energy-conserving reaction (12-13, 29, 31). Malate is an intermediate in this process (FIG. 1A) but requires only a single reducing equivalent for synthesis from phosphoenolpyruvate (PEP). A homo-malate fermentation could produce 2 moles of malate per mole of glucose at redox balance, preserve all glucose carbon, and incorporate two additional molecules of CO2 with a product yield of 149% that of glucose (weight basis). Thus, a need exists for providing genetically engineered microorganisms suitable for the production of malate.